Animal treatment

ABSTRACT

A method for affecting a physiological response of an animal to circulating level of prolactin and/or prolactin mimetics, characterised by the step of a) modulating prolactin receptors.

TECHNICAL FIELD

The present invention relates to a method of animal treatment.

BACKGROUND ART

The understanding of an animal's physiological processes has long been agoal of researchers and the agricultural industry.

A better understanding allows the development of better agriculturalpractices, improving productivity, farm management and profitability.

With a greater understanding comes the ability to manipulate an animal'sphysiological processes, further boosting productivity andprofitability.

Prolactin (PRL), a hormone of the anterior pituitary whose secretionvaries seasonally in many species (Table 1), is involved in thephysiological regulation of growth and development, hair and woolgrowth, reproduction, water and electrolyte balance, metabolism,behaviour and immune function [Bole-Feysot et al., 1998, Goffin et al.,2002].

For example, prolactin has previously been implicated in the control ofhair growth in various species [Lincoln, 1989] including wool growthcycles in primitive and shedding breeds of sheep [Lincoln, 1990; Lincolnand Ebling, 1985].

More recently, prolactin receptors (PRLR) have been identified in thewool follicle [Choy et al., 1997] revealing a physiological mechanismwhereby circulating prolactin can mediate wool growth cycles. However,to date the effects of prolactin on wool and hair growth in seasonalmammals, and particularly in modern non-shedding sheep breeds, are notwell understood.

It would be desirable to more fully understand this relationship and toprovide methods of manipulating prolactin dependent processes including,but not limited to, lactation, fertility and meat yields.

Prolactin is a peptide hormone comprising approximately 200 amino acidsand a molecular weight of 23,000 kDa [Freeman et al., 2000]. Circulatingprolactin is synthesised and released by specialised pituitary cellscalled lactotrophs, under the control of hypothalamic factors [Freemanet al., 2000].

Prolactin is also reported to be synthesised in an increasing number ofextra-pituitary tissues allowing for local autocrine and paracrineeffects [Wu et al., 1995; Craven et al., 2001]. It is thought to beresponsible for as many as 300 different effects on central andperipheral tissues [Bole-Feysot et al., 1998; Goffin et al., 2002].

Prolactin Secretion

Pituitary prolactin secretion is influenced by physiological factorsincluding photoperiod, temperature, pregnancy, parturition and lactationand stress.

Photoperiod: Prolactin secretion varies seasonally in many species(Table 1) being higher in long days (summer) than in short days(winter).

Temperature: A direct effect of the ambient temperature on prolactinconcentration has been observed in cattle [Wettermann and Tucker, 1974;Wettermann et al., 1982] and goats [Prandi et al., 1988] with increasingprolactin secretion with rising temperatures.

Pregnancy and lactation: During pregnancy the maternal pituitaryincreases in size, primarily as a result of hyperplasia and hypertrophyof lactotrophic cells [Djiane and Kelly, 1993]. Maternal plasmaprolactin levels remain low throughout most of the gestational periodbut increase rapidly in late term to reach maximal levels around thetime of parturition.

In sheep, most studies [Fitzgerald et al., 1981; Kelly et al., 1974;Kendall, 1999; Lamming et al., 1974] show that maternal prolactinconcentrations range between 10 ng/mL and 50 ng/mL during the first 100days of gestation. These levels are comparable to or lower than basalprolactin concentrations in non-pregnant ewes over the same period[Fitzgerald et al., 1981, Kendall, 1999).

A rise in maternal prolactin concentration is usually observed a fewdays before parturition [Kelly et al., 1974; Lamming et al., 1974,Kendall, 1999]. During the final stages of labour and at parturition,rapid pulses of prolactin are released, levels reaching betweenapproximately 100 ng/mL and 700 ng/mL [Kelly et al., 1974; Kendall,1999; Lamming et al., 1974; Peterson et al., 1990].

Lactation is also associated with raised plasma prolactin. In sheep,basal prolactin concentrations throughout early lactation range from100-150 ng/mL, however suckling causes a rise in prolactin levels to ashigh as 800 ng/mL [Kendall, 1999; Lamming et al., 1974].

By mid-lactation, basal levels are 20-100 ng/mL and suckling causes arise in prolactin concentration from 20 ng/mL to up to 400 ng/mL. Plasmaprolactin concentrations decline as lactation advances [Kendall, 1999;Lamming et al., 1974] and declines further after weaning [Rhind et al.,1980].

Prolactin Receptors

Prolactin has been shown to bind to specific high affinity cell surfacereceptors.

Signal transduction via these receptors initiates a cascade oftissue-specific gene transcription and translation resulting inphysiological adaptations [Freeman et al., 2000].

Prolactin receptors have been identified in wide variety of tissuesincluding the liver, uterus, mammary gland, kidney and skin [Barash etal., 1983; Cassy et al., 1999; Choy et al., 1997].

Multiple forms of prolactin receptor can arise by alternative splicingof a single gene [Ormandy et al., 1998; Bole-Feysot et al., 1998]. Twotypes of mRNA encoding a long and a short form of the prolactin receptorare detected in ovine and bovine tissues [Anthony et al., 1995]. Whilethe specific functions of the variant proteins are uncertain it ispresently thought that the major physiological effects of prolactin areexerted through the long form of the receptor.

The predominant form of prolactin has two receptor binding sites andcomplexes first with one receptor molecule to form a dimer, and thentransiently with a second receptor allowing the two receptors and theirauxiliary signalling molecules to interact [Gertler et al., 1996].Signal transduction only proceeds when the receptor-hormone-receptortrimeric complex is formed (FIG. 10).

The in vivo regulation of prolactin receptors is complex and variesbetween tissues. Changing steady state levels are dependent on therelative rates of synthesis, internalisation and recycling of receptors[Barash et al., 1983; Barash et al., 1986; Posner et al., 1975].

Cellular distribution and abundance of prolactin receptor mRNA issimilar to the distribution and abundance of prolactin receptor proteinacross a range of fetal and adult tissues [Freemark et al., 1993;Maaskant et al., 1996; Royster et al., 1993]. In skin for example,epithelial cells in resting hair follicles show higher immunoreactivity[Choy et al., 1997] and in situ hybridisation signal [Nixon et al.,2002] than in growing follicles. These histochemical studies support theproposition that prolactin receptor is largely transcriptionallyregulated, even though the translated products can vary in size from 30to 95 KDa Maaskant et al., 1996].

Short-term up-regulation by prolactin of its own receptors has beenreported in some tissues and tissue explants [Barash et al., 1986;Posner et al., 1975; Rui et al., 1986].

Both up- and down regulation have also been observed, dependent on thedose of prolactin [Rui et al., 1986].

Physiological Effects of Prolactin

Lactation

The increase in prolactin at the time of parturition stimulates thefinal phases of lactatogenesis and is essential to normal milkproduction in the subsequent lactation [Ostrom, 1990]. Failure or adelay in onset of lactation occurs when the periparturient prolactinsurge is abolished using bromocriptine (an inhibitor of prolactinsecretion) in ewes [Fulkerson et al., 1975; Peterson et al., 1991;Peterson et al., 1997; Schams et al., 1984], cows [Akers et al., 1981;Peel et al., 1978], and goats [Forsyth and Lee, 1993]. Infusions ofexogenous bovine prolactin can prevent bromocriptine-induced reductionsin milk yield in cows [Akers et al., 1981].

Milk yields are lower in bromocriptine treated ewes than in control ewesover the first 4 weeks of lactation [Peterson et al., 1997], while milkprotein content is increased. In rabbits, milk yields during anestablished lactation can be reduced for up to 36 h following a singlebromocriptine injection [Mena et al., 1982]. Recovery in milk yield inbromocriptine-treated rabbits is accelerated by single injection of 3 mgof prolactin.

Pregnant dairy heifers or ewes exposed to a long day photoperiod priorto parturition have a significantly larger periparturient prolactinsurge relative to those in normal or short day photoperiods [Kendall,1999; Newbold et al., 1991]. In addition to the well-characterisedincrease in milk yield of a long-day photoperiod during an establishedlactation [Dahl et al., 1997; Peters et al., 1978] there is evidencethat altered photoperiod during the dry period may affect milk yieldduring the subsequent lactation [Petitclerc et al., 1998].

Colostrum is defined in the dairy industry as the first milks followingcalving that have more than 1.45 g/L immunoglobulin G1. Normallycolostrum ceases being produced within 4-5 days of calving when copiousmilk production begins. Administration of bromocriptine to cows, toblock the surge in prolactin at calving, both delays the onset of milkproduction following calving [Akers et al., 1981] and concentratesimmunoglobulin G1 in the milk out past a week [Johke and Hodate, 1983].More recent work shows that prolactin directly down-regulates theimmunoglobulin G1 receptor, which transports immunoglobulin G1 intocolostrum, in mammary tissue [Barrington et al., 1997].

During the bovine lactation there are also variations in milkcomposition that are dependent on time of year rather than stage oflactation or nutritional status [Auldist et al., 1998] suggesting aphotoperiodic mechanism.

Fibre Growth

Many mammals, including sheep, exhibit seasonal pelage growth [Ling,1970] that is entrained by photoperiod and mediated by prolactin[Lincoln, 1990; Rougeot et al., 1984].

For example, long-woolled sheep breeds have high wool growth rates insummer and low growth rates in winter which is reflected in theassociated variation in fibre diameter and length growth rate [Sumnerand Revfeim, 1973; Woods and Orwin, 1988].

Increased variability in diameter along the fibre is, in turn,associated with a reduction in fibre tensile strength. The consequencesof this inconsistency, in conjunction with the reduced productivity,extend from farm management and profitability to wool processing andmarketing.

While the seasonal growth pattern is driven largely by changes indaylength, the fall in winter wool growth coincides with apregnancy-induced decline in follicle output [Pearson et al., 1999b] andwith reduced pasture availability. The reductions in follicle outputduring winter and during pregnancy can be ameliorated, but notprevented, by nutritional management [Hawker et al., 1984; Masters etal., 1993; Oddy, 1985] suggesting that hormonal mechanisms may beinvolved.

Growth and Development

Long days increase growth rates in cattle and accelerate the onset ofpuberty [Peters et al., 1980]. An effect on nutrient partitioning(increased feed efficiency and lean gain) has also been linked withincreased levels of prolactin in cattle and sheep [Schanbacher andCrouse, 1980; Tucker et al., 1984]. There is some direct evidence thatprolactin may be anabolic in a number of species [Nicoll, 1980]. Theinfusion of prolactin increases nitrogen retention in sheep held indarkness [Brinklow and Forbes, 1983], while immunising sheep againstprolactin suppresses body growth rates [Ohlson et al., 1981].

Reproduction

Effects of daylength and of prolactin on reproduction are wellcharacterised in many species [Curlewis, 1992; Loudon and Brinklow,1990; Reiter, 1980; Smith et al., 1987; Soares et al., 1991]. Inseasonal breeders, the onset of the breeding season and of puberty canbe controlled by manipulation of the photoperiodic environment [Hansen,1985] and of circulating prolactin [Loudon and Brinklow, 1990; Smith etal., 1987]. In species with an obligate seasonal embryonic diapause(e.g. mink), the seasonal increase in prolactin can have a luteotrophicor luteostatic actions [Curlewis, 1992].

Prolactin also influences the reproductive axis in males. For example,suppression of prolactin in rams with bromocriptine in summer decreasessteroidogenic and spermatogenic activity in the testes [Regisford andKatz, 1993; Regisford and Katz, 1994] and causes regression of accessorysex glands [Barenton and Pelletier, 1980].

All references, including any patents or patent applications cited inthis specification are hereby incorporated by reference. No admission ismade that any reference constitutes prior art. The discussion of thereferences states what their authors assert, and the inventors reservethe right to challenge the accuracy and pertinency of the citeddocuments. It will be clearly understood that, although a number ofprior art publications are referred to herein, this reference does notconstitute an admission that any of these documents form part of thecommon general knowledge in the art, in New Zealand or in any othercountry.

It is an object of the present invention to address the foregoingproblems or at least to provide the public with a useful choice.

Further aspects and advantages of the present invention will becomeapparent from the ensuing description which is given by way of exampleonly.

DISCLOSURE OF INVENTION

According to one aspect of the present invention there is provided amethod for affecting a physiological response of an animal tocirculating level of prolactin and/or prolactin mimetics, characterisedby the step of modulating prolactin receptors.

The term ‘physiological response’ should be taken to mean anyphysiological response of an animal that arises directly or indirectlyas a result of modulating prolactin receptors. This can includephysiological processes such as lactation, hair and wool growth, muscledevelopment and/or fertility. It should be appreciated that these aregiven by way of example only and should not be viewed as limiting in anyway, for prolactin is thought to be responsible for as many as 300different effects on central and peripheral tissues.

The term ‘circulating level’ should be taken to mean the concentrationof prolactin circulating in the blood of an animal.

The term ‘prolactin mimetic’ should be taken to mean a molecule whichbecause of its structural properties is capable of mimicking thebiological function of prolactin, such as causing prolactin receptorsignalling or altering the sensitivity or number of prolactin receptors.

The term ‘prolactin receptor(s)’ refers to any type of receptor to whichprolactin is known to bind.

The term ‘modulation’ should be taken to mean the artificialinterference on prolactin receptors by a number of means. This mayinclude treatments which either alter the receptor number and/or alterthe sensitivity of the receptors to prolactin.

For example, modulation may have a number of effects on prolactinreceptors, such as changes in the regulation of prolactin receptortranscription, mRNA stability and translation, or receptor sensitivity.However, this should not be seen as limiting and it should beappreciated that receptor modulation could include a range of othereffects.

The modulation of prolactin receptors may be brought about by a numberof means. Preferably, the modulation of receptor numbers is broughtabout by a sustained increase in the initial circulating level ofprolactin and/or prolactin mimetics, followed by the decrease back tonormal or low levels.

The modulation of prolactin receptors is thought to primeprolactin-dependent physiological processes to a second increase inprolactin levels. This second increase in prolactin may be brought aboutnaturally, for example by an increase in the photoperiod to which ananimal is exposed, or may be artificially induced in an animal.

Prolactin is preferably administered by any method which will induce asustained increase in circulating prolactin, such as by intravenousinfusion, by a slow release bolus or implant. However, this should notbe seen as limiting and a number of methods known in the art could beused.

The administration may be carried out to effect the initial modulationof the receptors and/or for the later introduction of a second event ofelevated prolactin that the modulated receptors respond to.

The inventors have surprisingly found that in order to effect themodulation of physiological processes, the elevated circulatingprolactin levels must be sustained, prior to the reduction to normal orlow levels after infusion. Merely providing daily injections ofexogenous prolactin were not sufficient to alter the response of ananimal.

After the temporary increase, the circulating levels of prolactin arepreferably reduced to normal or low circulating levels. This reductionmay be brought about by either decreasing the photoperiod to which theanimal is exposed and/or reducing or terminating the exogenousadministration of prolactin or prolactin mimetics. The inventors havefound that this profile upregulates the expression of the prolactinreceptor gene over a sustained period of time.

In one preferred embodiment of the present invention, the photoperiod,i.e. duration of daylight to which animals are exposed, has been foundto have an effect on circulating prolactin levels and thus also onprolactin receptors themselves.

Normal (ND) photoperiod may be defined as the seasonally varying naturaldaylength. Long day (LD) photoperiod may be defined as 16 h light and 8h dark; and short day (SD) photoperiod may be defined as 8 h light and16 h dark.

In preferred embodiments, the photoperiod may be altered in a controlledenvironment by any of the methods well known in the art.

In other preferred embodiments of the present invention theadministration of exogenous prolactin may be given to animals to mimicthe effect of increased photoperiod (LD) on the circulating levels ofprolactin.

Exogenous purified prolactin from any commercially available source, forexample a recombinant product or from a protein extract derived fromsheep pituitary glands, is preferably given over an extended period oftime.

For example, to alter hair and wool growth in sheep, the inventors havefound that the optimum length of sustained prolactin increase is from3-18 days, more preferably 3-15 days and most preferably 9 days. Whileit is expected that the effect on hair and wool growth may still occurwhen the increase in circulating prolactin is sustained for more than 18days, it is unlikely that such lengthy treatments would be particularlycost effective.

In this example, the concentration of circulating prolactin ispreferably first increased by 5 ng/mL-800 ng/mL above normal levels andthen returned to normal levels.

Most preferably, the concentration is increased by 5 ng/mL to 200 ng/mLabove normal levels.

When endogenous circulating levels of prolactin are low (<50 ng/ml),small increases in prolactin of less than 5 ng/ml may also be effectivein inducing changes in prolactin receptor abundance or sensitivity.

It should be appreciated that this is given by way of example only andthe lengths of prolactin increase and the concentration thereof shouldnot be seen as a limitation on the present invention in any way. Otherspecies and other tissues are expected to require different optimalconditions.

In another preferred embodiment of the present invention the increase inthe circulating level of prolactin may be brought about by theincorporation into the animal's genome of an inducible recombinantnucleotide sequence encoding biologically active prolactin or arecombinant nucleotide sequence encoding a molecule which enhancesendogenous prolactin activity. By over expressing prolactin, the timingand level of expression of specific genes may be altered in transgenicanimals.

For example, the prolactin gene sequence could be inserted into aninducible gene cassette under the control of a suitable mammary-specificpromoter such as a milk protein, a promoter that expresses in all celltypes (constitutive expression), or the prolactin promoter and/or asuitable enhancer sequence to drive transcription thereof.

This cassette would also preferably contain 3′ flanking DNA that couldstabilise the mRNA and may contain downstream regulatory sequences.

This DNA cassette could be introduced into the genome of an animal bymicroinjection of the DNA into pronuclei of eggs (described byL'Huillier et al., 1996) which are subsequently transferred back torecipient animals and allowed to develop to term. This technique for theproduction of transgenic animals is described by Hogan et al. (1994).

Another way to produce transgenic animals involves transfection of cellsin culture that are derived from an embryo, or foetal or adult tissuesfollowed by nuclear transfer and embryo transfer to recipient animals.Alternatively, the gene cassette may be bound to mammalian sperm anddelivered to the egg via in vitro or in vivo fertilisation to produce anon-human transgenic animal.

Manipulation of the developmental regulation or the level of expressionof prolactin may be used to alter the characteristics of thephysiological responses of an animal, or alter the rate whereby theseoccur.

Alternatively, the gene cassette may comprise a DNA sequence encoding amolecule which enhances endogenous prolactin activity or alters thesecretion of substances affecting prolactin release or plasma half-lifesuch as prolactin binding proteins, oestrogen, GnRH associated prolactininhibiting factor, pit-1, hypothalamic dopamine, serotonin and gammaaminobutyric acid or any other suitable molecule as would be known to aperson skilled in the art [Freeman et al., 2000].

According to another aspect of the present invention there is provided amethod of modulating prolactin receptors by artificially increasing thecirculating level of prolactin to a concentration and for a period oftime as required, followed by the reduction in the circulating level ofprolactin to basal or lower levels.

Preferably, the circulating levels of prolactin are reduced, after thetemporary sustained increase, to normal or low circulating levels. Thisreduction may be brought about by either decreasing the photoperiod towhich the animal is exposed, and/or reducing or terminating theexogenous administration of prolactin or prolactin mimetics. Theinventors have surprisingly discovered that the reduction in circulatingprolactin back to basal or normal levels is important for the modulationof wool growth.

In animals which have an inducible recombinant nucleotide sequenceincorporated into their genome which increases prolactin either directlyor indirectly, the circulating levels of prolactin may be reduced by theadministration of an inhibitor of prolactin synthesis or by cessation ofprolactin induction.

In transgenic animals, the induced increase in circulating levels ofprolactin may be reduced after a desired period by switching off theinducible gene cassette, and the effects on the physiological processesof an animal measured by known methods.

In another preferred embodiment of the present invention, the modulationof prolactin receptors may be achieved by the administration ofantibodies capable of affecting the response of the prolactin receptorsto circulating prolactin levels. These antibodies may have stimulatoryor inhibitory effects on prolactin receptors, act as prolactin mimetics,or may bind to circulating prolactin/prolactin mimetic molecules to keepthese in circulation for longer, prolonging the physiological response.

Immunological manipulations of this type of hormone/receptor system canbe applied to animal production (Aston et al., 1991; Pell & Aston,1995). Inhibitory and stimulatory antibodies to rabbit prolactinreceptor have been described (Djiane et al., 1985) as have antibodiesthat enhance the activity of growth hormone (Holder et al., 1985; Beatie& Holder, 1994) and insulin-like growth factor-I (Hill & Pell, 1998).Again the timing, duration and effective increase and decrease inprolactin-like activity are as described above.

The modulation of prolactin receptors may be induced at any timethroughout the year to affect physiological processes of an animal,although it may preferably be given at times when natural circulatingprolactin levels are low such as during the winter. It will beunderstood by a person of skill in the art that the prolactin profile ofa species of interest would be of use in deciding the optimum times inwhich to carry out the method of the present invention.

Knowledge of the prolactin profiles of an animal of interest would beuseful in carrying out the method of the invention at the preferredtiming, i.e. when natural circulating levels of prolactin are notchanging rapidly and it is within the capacity of a person skilled inthe art to obtain such a prolactin profile.

However, the inventors have shown that the method of the invention willwork even when the natural circulating level of prolactin is high, forexample during parturition, so that timing of the modulation ofprolactin receptors does not appear to be restrictive. The method of theinvention is expected to work throughout the year and not be dependenton the seasonal or pregnancy-induced changes in the prolactin profile ofan animal, though these changes can be used to affect the physiologicalresponse of an animal after the initial modulation of prolactinreceptors.

The present invention also provides an animal treated by the method ofthe invention including transgenic animals and their off-spring.

According to a further aspect, the invention provides animal productsproduced by an animal treated by the method of the invention.

The inventors have also devised a model to predict the effect of aparticular treatment as a guideline for developing the best method fortreatment or of experimental design.

Using this model, it is possible to determine parameters for aparticular species or tissue and to design the optimal timing, plasmaprofile and dosage of a temporary but sustained prolactin (or a mimetic)treatment or immunological manipulation to alter short and long-termphysiological responses to these.

The model can also be used to predict the appropriate timing, plasmaprofile and dosages of single or serial, temporary sustained prolactin(or a mimetic) treatment(s) or immunological manipulation so as tomodulate prolactin receptors to optimally enhance responses to prolactinor to prolactin mimetics.

In other embodiments, the model may be used to design the appropriatetiming, plasma profile and dosages of single or serial, temporarysustained prolactin (or a mimetic) treatment(s) or immunologicalmanipulation so as to modulate prolactin receptors to optimally inhibitresponses to prolactin or to prolactin mimetics.

It should be appreciated that other models may be developed which canpredict changes in receptor numbers, receptor sensitivity and/or otherreceptor parameters.

Specifically, the inventors have devised a mathematical model ofprolactin-prolactin receptor interaction, developed from knowledge ofreceptor dynamics in general and prolactin receptor biochemistry inparticular. By using experimental data showing long-term prolactinreceptor gene transcription induced by circulating prolactin, a numberof experimentally observed phenomena were predicted.

Firstly, the necessity for a rise, a sustained elevated concentrationand then a decline in circulating prolactin concentration for optimalshort-term biological effects and for longer term stimulatory effects onprolactin receptor numbers (and therefore enhanced biologicalresponsiveness to prolactin or prolactin mimetics) were predicted. Inaddition, the ineffectiveness of prolactin injections versus theenhanced responses to sustained prolactin infusions on prolactinreceptor numbers was also predicted.

Another experimentally observed effect predicted by the inventors' modelwas that a long term depression of prolactin receptor numbers andtherefore diminished biological responsiveness to prolactin or prolactinmimetics resulted in response to very high or prolonged prolactin (orprolactin mimetic) treatment.

DESCRIPTION OF THE DRAWINGS

The invention will be further described by reference to the figures ofthe accompanying drawings (pages 1/19-19/19) in which:

FIG. 1 shows the experimental design for the six trials using sheepdisclosed herein. The length of the bars represents the duration of eachtreatment as shown in the legend; SD means short days; ND means naturaldays; LD means long days. NP means non-pregnant (dry) ewes; L meansbreeding (lambed) ewes; BrB means bromocriptine administered to breedingewes before parturition; BrA means bromocriptine administered tobreeding ewes after parturition; PRL-INF means prolactin administeredintravenously and PRL-INJ means prolactin injected subcutaneously;

FIG. 2 shows the effect of photoperiod manipulation on circulatingprolactin and prolactin receptor expression for Trial 1. The change fromshort- to long-day photoperiod (SD:LD) on 15 January (SouthernHemisphere summer) is indicated by an arrow. Top panel: mean plasmaprolactin concentrations measured by radioimmunoassay from morningsamples: (▪) values for control animals exposed to natural day length,(♦) values for light-treated animals. Bars show the standard error ofthe mean. Bottom panel: relative abundance of mRNA for long form (●) andshort form (▾) of PRLR determined by RNase protection assay from animalssacrificed throughout the experiment. Lines follow averages of duplicateanimals.

FIG. 3 shows the midside mean fibre diameter (top panel), midside cleanwool growth rate (second panel), plasma prolactin and midside clean woolgrowth rate for selected groups (third and fourth panels) and the meanclean fleece weight (±standard error of the mean) collected at shearing(bottom panel) for each treatment group of Trial 2. Key: (□) NDnon-pregnant; (▪) ND-lambed; (▴) ND-BrB; (▾) ND-BrA; (◯) LDnon-pregnant; and (●) LD-lambed. Bottom panel: □ ND non-pregnant; ▪ND-lambed;

ND-BrB;

ND-BrA;

LD non-pregnant; and

LD-lambed ewes. The vertical error bar in the first and second panelsshows the pooled SED of the means.

FIG. 4 shows the mean plasma prolactin concentration of the treatmentgroups of Trial 3 for (●) LD-lambed, (▪) ND-lambed and (□) NDnon-pregnant ewes (top panel); (♦) PRL-INJ ewes (middle panel) and (▴)PRL-INF ewes (bottom panel). Prolactin administration was for 18 daysindicated by the hatched bar.

FIG. 5 shows for each of the treatment groups of Trial 3, the mean fibrediameter (top panel) and the mean clean wool growth rates (middle panel)(Key: (●) LD-lambed; (♦) PRL-INJ; (▴) PRL-INF; (▪) ND-lambed and (□) NDnon-pregnant ewes); and the mean clean fleece weight (±standard error ofthe mean) collected at shearing (bottom panel) (Key: □ ND non-pregnant;▪ ND-lambed;

PRL-INF;

PRL-INJ and

LD-lambed ewes). Prolactin administration was for 18 days indicated bythe hatched bar.

FIG. 6 shows for each of the treatment groups of Trial 3, the meanrelative skin PRLR (long form) mRNA expression measured by real-timePCR. Prolactin administration was for 18 days indicated by the hatchedbar. Key: (◯) non-pregnant ewes, (♦) pregnant ewes, (▴) infused pregnantewes, (●) injected pregnant ewes and (▪) are LD pregnant ewes. Verticalbars show SED between means for each sampling date.

FIG. 7 shows the mean plasma prolactin concentration of the treatmentgroups of Trial 4 for (□) ND non-pregnant ewes (top panel); (▪) 3-dayPRL infusion (second panel) (♦) 9-day PRL infusion (third panel) and (▾)18-day PRL infusion ewes (bottom panel). Prolactin administration wasfor the periods indicated by the hatched bars. P represents the meandate of parturition; W represents the date of weaning;

FIG. 8 shows for each of the treatment groups of Trial 4, the mean fibrediameter (top panel) and the mean patch clean wool growth rates (middlepanel) (Key: (▪) ND-lambed—estimated from non-pregnant ewes; (♦) 3-dayPRL infusion; (●) 9-day PRL infusion; (▾) 18-day PRL infusion); and themean total clean patch weight (±standard error of the mean) collectedover the trial (bottom panel) (Key: ▪ ND-lambed—estimated fromnon-pregnant ewes;

3-day PRL infusion;

9-day PRL infusion and

18-day PRL infusion ewes). Prolactin administration was for the periodsindicated by the hatched bars.

FIG. 9 shows for each of the treatment groups of Trial 4, the meanrelative skin PRLR (long form) mRNA expression measured by real-timePCR. Prolactin administration was for 3, 9 and 18 days indicated by thehatched bars. Key: (▪) pregnant ewes, (Δ) 3-day infused pregnant ewes,(δ) 9-day infused pregnant ewes and (◯) 18-day infused pregnant ewes.Vertical bars show SED between means for each sampling date.

FIG. 10 shows the mean plasma prolactin concentrations (top panel) andthe relative log prolactin receptor mRNA concentrations (bottom panel)of the treatment groups of Trial 5: (▪) ND ewes; (◯) SD saline infused;and (♦) SD prolactin infused ewes. The vertical error bars in the secondpanel show SED between means for each sampling date. Prolactinadministration was for the periods indicated by the hatched bars.

FIG. 11 shows the mean plasma prolactin concentrations (top panel) andthe relative log prolactin receptor mRNA concentrations (bottom panel)of the treatment groups in Trial 6. Key: (Δ) SD Romney ewes; (♦) SDprolactin infused Romney ewes; (◯) SD Wiltshire ewes; and (▪) SDprolactin infused Wiltshire ewes. The vertical error bars in the secondpanel show SED between means for each sampling date. Prolactinadministration was for the periods indicated by the hatched bars.

FIG. 12 shows the relative log prolactin receptor mRNA concentrations inthe liver, mammary gland and skin of rabbits in Trial 7 before and aftera 7-day infusion of ovine prolactin.

FIG. 13 shows for each of the treatment groups of Trial 8 the meanplasma prolactin concentrations (top panel) and the relative logprolactin receptor mRNA concentrations (bottom panel) (±standard errorof the mean). Key: (◯) saline and bromocriptine control group; 3-dayprolactin infusion group (▴); and 7-day prolactin infusion group (▪).

FIG. 14 shows the schematic structure of a mathematical model ofprolactin receptor regulation by prolactin. The four output variablesare the concentration of plasma prolactin, and the numbers of unbound,and bound receptors as dimeric and trimeric complexes (shown in boxes).The number of bound receptors increases due to association of prolactinwith unbound receptors and decreases because of degradation (D) anddissociation back to the unbound state. The number of unbound receptorsdecreases because of binding and degradation (D), and increases due tosynthesis (S) and dissociation processes. The concentration of plasmaprolactin decreases mostly because clearance (D) whilst secretion andartificial prolactin input cause an increase. These statements areexpressed as a set of linked differential equations (see text).

FIG. 15 shows a mathematical simulation of the concentrations of unboundand total receptors (top panel, dashed and bold lines respectively),signalling trimer complex (middle panel), and concentration of plasmaprolactin (bottom panel) with a nine day infusion of prolactin startingat day 5.

Initial conditions are determined by equilibrium at assumed constantvalues and parameter settings. Because no prolactin is being infusedover the initial ten days, the plasma prolactin concentration and thenumber of bound and unbound receptors do not change. The number ofunbound receptors drops as the prolactin is infused, with acorresponding rise in bound receptor number. Once the infusion isswitched off, the number of bound receptors declines back to the initialequilibrium, and the number of unbound receptors reaches its highestlevel, then slowly relaxes to the equilibrium level.

FIG. 16 shows a mathematical simulation of the concentrations of unboundand total receptors (top panel, dashed and bold lines respectively),signalling trimer complex B₂ (middle panel), and concentration of plasmaprolactin P (bottom panel) with a nine day infusion of prolactin.Conditions are as for FIG. 15, except that the prolactin pulse occursafter 17 days. The changes in level of receptors are independent of thetiming of the pulse of prolactin.

FIG. 17 shows a mathematical simulation of the numbers of unbound andtotal receptors (top panel, dashed and bold lines respectively), boundreceptors in signalling trimer complex B2 (middle panel), andconcentration of plasma prolactin P (bottom panel) with two successivenine-day infusions of prolactin. The first infusion alters theconditions for the second infusion resulting in higher levels of boundreceptors after the second infusion.

FIG. 18 shows a mathematical simulation of the numbers of unbound andtotal receptors (top panel, dashed and bold lines respectively) andbound receptors in signalling trimer complex B2 (middle panel) inresponse to a series of prolactin injections followed by a nine-dayinfusion of prolactin. Prolactin concentration is shown in bottom panel.Injections cause elevations of prolactin which are greater than thatcaused by infusion, but with much smaller duration, and result in acomparatively smaller response, showing the system is not sensitive toshort-term increases in prolactin.

FIG. 19 shows that the peak trimer concentration, and therefore thepotential biological response, resulting from a 9-day infusion ofprolactin varies in a non-linear fashion with the elevation of prolactinover its basal level. There is an optimal infusion level for anyinfusion duration, indicated by the peak, after which signalling levelstarts to decrease.

DETAILED DESCRIPTION OF THE INVETION

As defined above, the present invention is directed to affecting aphysiological response of an animal to circulating levels of prolactinand/or prolactin mimetics by modulating prolactin receptors.

The invention is based upon the inventors' investigation into theprofiling of prolactin, the effect of prolactin upon prolactin receptorsand the development of a model to predict these effects and allow bettermethods of treatment and experimental design.

Non-limiting examples of the invention will now be provided.

Protocol

Eight trials are described to demonstrate the principles of theinvention by describing the effects of a temporary sustained prolactinsurge on short and long term prolactin receptor gene expression, and byexperimentally manipulating plasma prolactin profiles in sheep causingchanges in annual wool growth patterns.

Two breeds of sheep were utilised and showed differing effects relatedto their wool growth characteristics. The New Zealand Romney grows longrelatively course wool continuously, but with marked seasonal variationin growth rate. The research conducted by the inventors showed thatprolactin modulates the seasonal production pattern of this breed. Bycontrast, the New Zealand Wiltshire is a meat breed with slow,discontinuous fleece growth. In these experiments, prolactin treatmentsaltered the timing of hair cycles and fleece shedding. Romney sheep wereused in trials 2 to 6. Wiltshire sheep were used in trials 1 and 6. Thedesigns for these trials are shown in FIG. 1.

The research was carried out in the Southern Hemisphere where summer,autumn, winter and spring correspond to the calendar monthsDecember-February, March-May, June-August and September-Novemberrespectively. The principal objectives were to:

-   -   to determine the effects of a temporary sustained prolactin        surge (induced by long day photoperiod) on the regulation of        prolactin receptor gene transcription (Trial 1);    -   to determine the effect of differing profiles of endogenous        prolactin on wool growth (Trial 2);    -   to determine the effect of a sustained, temporary increase in        the plasma concentration of prolactin, induced by infusion of        exogenous prolactin, on subsequent wool growth patterns (Trials        3 and 4);    -   to determine the effects of two different modes of        administration (injection versus infusion) on subsequent wool        growth (Trial 3);    -   to show that both stimulatory and inhibitory wool growth effects        can be achieved depending on the dose and mode of administration        (Trials 3 and 4).    -   to show that two successive periods of exogenous prolactin        administration to non-pregnant ewes can perturb prolactin        receptor gene expression on each occasion (Trial 5).    -   to show that prolactin effects on prolactin receptor gene        expression depend on genotype (Trial 6).    -   to show that prolactin administration can perturb prolactin        receptor gene expression in other species apart from sheep and        in other tissues apart from skin (Trials 7 and 8).

For Trials 2-6 and 8 the data relating to the wool growth measurements,plasma prolactin concentrations and prolactin receptor gene expressionwere subjected to an analysis of variance at each sampling time to testthe effects of treatment. Plasma prolactin and prolactin receptor geneexpression values were log transformed before analysis to allowassumption of homogeneous variance in all experimental groups.

For the wool growth data, initial values were used as covariates foranalysis.

Trial 1

Long-Term Regulation of Prolactin Receptor Gene Transcription byProlactin

Wool follicle cycles were synchronised in New Zealand Wiltshire sheepusing an artificial photoperiod regime to manipulate circulatingprolactin, as previously described [Parry et al., 1996].

Twenty-nine mature sheep (18 rams and 11 ewes) were maintained indoorson a constant diet of sheep pellets and hay for six months from 11October (Southern Hemisphere spring). The animals were allocated to oneof two groups. Group 1 (n=9; 4 rams and 5 ewes) was exposed to normaldaylight via windows. Group 2 (n=20; 14 rams and 6 ewes) were exposed toa constant short day length (L8:D16) for 13 weeks and then, from 15January (day 0), to long day length (L16:D8) until 23 April (day 98).

Such an artificial lighting regime has been shown to abolish the normalspring rise in pituitary prolactin secretion, then, with the photoperiodtransition in mid summer and release of prolactin suppression, tosynchronously induce follicle regression and interrupt wool growth[Nixon et al., 1997; Pearson et al., 1993].

Blood samples (5 ml) were collected from all animals by jugularvenipuncture at 2 to 10 day intervals from 22 October (85 days prior tothe change of photoperiod) until 22 April (day 97 after change ofphotoperiod). Prior to the change in photoperiod at day 0, blood sampleswere taken in the morning between 08:00 and 09:30. After day 0, bloodwas also collected in the evening between 20:00 and 21:30. Plasma wasseparated by centrifugation within 2 hours of blood collection.

Two control sheep from Group 1 were sacrificed on each of days 0, 28,and 98. Photoperiod treated sheep from Group 2 were killed over thecourse of the induced wool growth cycle; two on each of days 0, 7, 14,21, 28, 47 and 98. Samples of skin from the mid-sides of these animalswere frozen in liquid nitrogen and stored at −85 C. or fixed inphosphate buffered 10% formalin. Fixed skin was processed to paraffinwax and 7 μm transverse sections cut and stained by the Sacpic methodfor determination of follicle activity [Nixon, 1993]. Plasma prolactinconcentrations were measured in duplicate by radioimmunoassay aspreviously described [Nixon et al., 1993].

Ribonuclease Protection Assays

Total RNA was isolated from approximately Ig of each frozen skin samplecollected from Groups 1 and 2 by grinding to powder under liquidnitrogen in a freezer mill (SPEX 7700, Glen Creston Ltd, Middlesex, UK),and extracting with “TRIzol” reagent (Gibco BRL, Rockville, Md.)according to the manufacturer's instructions. RNA concentration wasmeasured by spectrophotometry at 260 nm and integrity verified on anagarose/formaldehyde gel.

Antisense riboprobes for ovine prolactin receptor [Anthony et al., 1995]and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Genbank accessionno. AF022183) were used in ribonuclease protection assays. The prolactinreceptor cDNA sequence spanned an alternatively spliced region in theproximal cytoplasmic domain and was therefore able to distinguish RNAvariants encoding long and short isoforms of prolactin receptorindicated by protected fragments of 441 bp and 549 bp respectively [Choyet al., 1997]. The GAPDH cDNA, encoding 424 bp of the 5′ region, wasgenerated by RT-PCR of sheep skin and cloned into pGemT vector (Promega,Madison, Wis.).

Both riboprobes were labelled with a-33P-uridine 5′-triphosphate(Amersham International, Buckinghamshire) by in vitro transcription fromlinearised plasmids using the Riboprobe Core System (Promega, Madison,Wis.).

RNase protection assays of both prolactin receptor and GAPDH werecarried out in duplicate using the Ambion RPAII Kit (Ambion, Austin,Tex.) following the manufacturer's instructions. Forty micrograms oftotal RNA was hybridised with both riboprobes at 45° C. overnight.Unhybridised RNA was removed by RNase digestion followed by inactivationof RNase and precipitation of protected fragments. These fragments wereseparated by electrophoresis on a 5% polyacrylamide/8M urea gel. Afterdrying, gels were exposed in intensifying screens to Kodak XAR film(Eastman Kodak, Rochester, N.Y.). Optical density of protected fragmentswas measured using Molecular Analyst Software (BioRad Laboratories,Hercules, Calif.) and prolactin receptor bands were standardised againstGAPDH measurements.

Results

Photoperiod Manipulation Altered Prolactin Secretion and Induced a WoolGrowth Cycle

In control animals exposed to normal changes in day length (Group 1),the maximum mean plasma prolactin concentration was observed on 6November (mean±SEM: 148±30 ng/ml) (data not shown). Prolactin levelsthen gradually declined over the experimental period, although somefluctuations occurred in association with animal management events andunusually high daytime summer temperatures. By comparison, circulatingprolactin was suppressed (P<0.001) in animals maintained in short daysduring spring (Group 2). Mean values at 15 days before the change inphotoperiod were 6±2 ng/ml and 7±1 ng/ml in Group 2.

Following transition into long days (day 0), prolactin levels increasedrapidly (FIG. 2). By 23 days after the change in photoperiod,concentrations were 81±12 ng/ml in Group 2. Peak evening prolactinlevels ranging from 134 ng/ml to 260 ng/ml were observed in individualanimals between 23 days and 70 days. Thereafter, plasma prolactinconcentration declined in all treated animals (FIG. 2).

Histological assessment of hair follicle growth status showed thatvirtually all follicles sampled from both treated and control animals atday 0 were in anagen (growth phase) (data not shown). Follicles remainedgrowing in control animals (Group 1) whereas follicles in light treatedanimals (Group 2) passed through a hair cycle.

By day 28, the majority of follicles sampled from animals undergoinglight treatment had entered telogen (resting phase) and in twoindividuals the percentage of growing follicles had reached a nadir of2%. The wool follicles progressively reactivated such that by day 98almost all had resumed growing.

Level of Prolactin Receptor Expression During a Prolactin Induced WoolFollicle Cycle

The abundance of prolactin receptor mRNA relative to GAPDH mRNA in theskin of Group 2 sheep varied following the photoperiod transition andconsequent rise and fall in plasma prolactin (FIG. 2). The samplingpoints covered the major divisions of the hair follicle cycle. Theinitial response, occurring between day 0 and day 7, was an apparentdecline in prolactin receptor mRNA. This corresponded to a smallincrease in plasma prolactin (P<0.01) of less than 10 ng/ml at five daysafter the photoperiod transition. No changes in skin or folliclemorphology were yet visible, but this time immediately preceded catagen.

From day 7 to day 47, prolactin receptor was up-regulated (long form:P<0.01) (FIG. 2). Over this period, there was a rapid and continuousincrease in plasma prolactin concentration and regression of folliclesto the telogen phase resulting in the shut down of fibre growth. Thetransition through catagen saw the most rapid changes in prolactinreceptor mRNA levels. Prolactin receptor mRNA was most abundant at day47 by which time hormone levels were about to fall.

By day 98, circulating prolactin had dropped and prolactin receptor mRNAapproached the levels observed at the start of the experiment whenfollicles were similarly in anagen. The relative abundance of prolactinreceptor mRNA in the skin of Group 1 (control) animals did notsignificantly differ from Group 2 animals when follicles were in anagen(day 0 and day 98) (data not shown).

In all RNase protection analyses, bands corresponding to prolactinreceptor long form protected fragment emitted more signal than those ofshort form protected fragments, indicating the greater abundance of longform transcripts. Both isoforms underwent a similar pattern of decreasefollowed by increase and return to anagen levels over theprolactin-induced cycle (FIG. 2). The ratio of long- to short form mRNAwas greater at 47 days when total prolactin receptor expression was at amaximum and the follicles were in proanagen, as compared with samples inanagen (P<0.05).

Trial 2

Effects of Differing Endogenous Prolactin Profiles on Wool Growth

Natural and experimental changes in plasma prolactin concentration andtheir effects on wool production in winter-lambing Romney ewes weremeasured.

Fourteen non-pregnant and 29 pregnant ewes were maintained indoors fromearly April 1995 for 12 months under controlled photoperiod andcontrolled dietary intake. Two groups (n=8) were held under long dayphotoperiod (16L:8D; LD non-pregnant and LD-lambed) while 2 others wereexposed to natural photoperiod (ND non-pregnant, n=6 and ND-lambed,n=7). Two further groups (n=7) of pregnant ewes housed in natural dayswere treated with bromocriptine, either from 1 week before parturition(ND-BrB) or 1-3 days after parturition (ND-BrA), to suppress prolactinsecretion.

Lambing occurred between 12 and 18 June. Plasma prolactin, mean fibrediameter and wool growth were measured at regular intervals and theresults were plotted in FIG. 3.

Results

Photoperiod and treatment with bromocriptine did not affect the birthweight or live weight changes of lambs. In ND non-pregnant ewes, changesin plasma prolactin concentrations were associated with seasonal changesin day length over the duration of the trial. In LD pregnant ewes,continued exposure to LD photoperiod from April caused a significantincrease in pre-parturition prolactin concentrations over the wintermonths compared to ewes held in normal days (24 vs. 11 ng/ml; P<0.02).

Prolactin concentrations were low in the ND-BrB group throughout thetrial, and in the ND-BrA group following the prolactin peak associatedwith parturition (data not shown). Apart from the ND-BrB group,prolactin concentrations in pregnant ewes increased rapidly a few daysprior to parturition and subsequently remained elevated. Prolactinconcentrations over parturition and lactation were highest in LD-lambedewes (FIG. 3).

Despite comparable maternal live weights, lambing was associated with alower clean fleece weight, and a reduced mean fibre diameter, staplelength and staple tensile strength compared to non-pregnant groups. LDewes grew significantly more clean wool than their ND counterparts(P<0.01) as a consequence of an increase in both mean fibre diameter andlength growth rate (FIG. 3). The larger prolactin surges associated withparturition and early lactation followed by a decline over mid tolate-lactation had a significant stimulatory effect on both wool growthrate (P<0.001) and fibre diameter (P<0.001) in LD-lambed ewes from Juneto September relative to ND-lambed ewes.

The complete absence of the peripartum prolactin surge was associatedwith longer-term inhibitory effects on wool growth rate. However,elevated plasma prolactin concentrations during pregnancy, and atparturition and early lactation, followed by a decline in prolactinlevels over mid to late-lactation, are linked to higher wool productionarising from increases in mean fibre diameter and fibre length growthrate.

Trial 3

Effects of Differing Prolactin Priming Profiles on Long-Term Wool Growthand Prolactin Receptor Gene Expression

The effects of two methods of administration of exogenous prolactin onwool growth were examined.

Forty-three mixed age Romney ewes were maintained indoors in individualpens from April until October, and fed to maintain a constant maternallive weight (independent of conceptus and fleece weights). A controlgroup (ND non-pregnant) was not mated (n=8). Thirty-five other ewes(comprising Groups 2-5) were mated in April. Groups 1-4 were maintainedin natural photoperiod while Group 5 (LD-lambed, n=10) was subjected tolong days (16L:8D) from 1 week after mating for the duration of thetrial (ie 18 days). Group 2 (ND-lambed, n=11) did not receive exogenousprolactin.

An ovine prolactin pituitary extract (0.80 mg/kg/day) was administeredby either daily subcutaneous injection to sheep of Group 3 (PRL-INJ,n=10) or by intravenous infusion via a jugular cannula to sheep of Group4 (PRL-INF, n=2) (from one week after mating for a temporary period of18 days).

The sheep were shorn on entry to the experiment and again at theconclusion of the experiment in late September and a mid-side woolsample was measured for fibre diameter and yield. Wool samples wereclipped from a standardised 100×100 mm mid-side patch on the right sideof each ewe at monthly intervals to determine changes in seasonal cleanwool production and mean fibre diameter. Wool growth and plasmaprolactin concentrations were measured at regular intervals and theresults were plotted in FIGS. 4 and 5.

Six midside skin biopsies were collected at intervals between April andJune for measurement of prolactin receptor long form gene expression byreal-time PCR and the results are plotted in FIG. 6. An assay for theshort form of the ovine prolactin receptor was also developed, butresults are not presented since only very low levels of expression weredetected.

Real-Time PCR Assay for Prolactin Receptor Long Form Gene Expression

The relative expression of prolactin receptor mRNA was measured usingTaqman real-time PCR (Applied Biosystems). RNA was extracted from skinsamples using TRIzol reagent according to manufactures protocol(Invitrogen). Total RNA was then quantified using RiboGreen reagent(Invitrogen) and all samples were standardized to a concentration of 0.5μg/μl of total RNA. The reverse transcriptase reaction (Superscript IIRT-PCR kit, Invitrogen) was used to generate single stranded cDNA from0.5 μg of total RNA.

The primers and probes for the long form of the prolactin receptor weredesigned using the Primer Express program (Applied Biosystems). Primerssequences were as shown in Table 2.

The 18S ribosomal RNA pre-developed assay reagent (Applied Biosystems)was used as an internal control and the reactions were set up accordingto the manufacturers instructions. Data was analysed using the softwareprovided by Applied Biosystems. Results on relative mRNA quantities wereobtained by the standard curve method.

Results

Of the 35 mated ewes, 2 did not produce a lamb and were excluded fromthe results. Lambing occurred between 9 and 19 September. Prolactinprofiles in the control and treated groups are shown in FIG. 4. Therewas no evidence that prolactin treatment had any adverse effect eitheron pregnancy maintenance or on the health and growth rate of the lambs(data not shown).

At the commencement of the experiment the clean wool growth rate did notdiffer between groups, however by September a strong treatment effectwas evident (FIG. 5; P<0.001). Clean wool growth rates declined in allgroups between April and May, indicative of the winter decline.

An effect of pregnancy was also reflected in the lower clean wool growthrates of mated ewes as compared to the non-pregnant group. Dailysubcutaneous injections of prolactin (0.8 mg/kg/day) had no initialeffect on the decline in clean wool growth rate compared to the pregnantcontrols. However, the September clean wool growth was lower (P<0.05) inPRL-INJ ewes than in untreated ND-lambed ewes.

The clean wool growth rate in the LD-lambed ewes did not differ fromND-lambed ewes but showed a tendency to rise more rapidly in August. BySeptember LD-lambed ewes had a significantly higher (P<0.025) clean woolgrowth rate than their ND-lambed controls. In contrast to othertreatment groups the decline in clean wool growth rate in the twoPRL-INF ewes was arrested in May and remained high (even above that ofnon-pregnant control ewes) throughout pregnancy before rising inSeptember to approximately twice that of untreated ND-lambed ewes(P<0.001).

At the commencement of the experiment the mean fibre diameter did notdiffer between groups, however by September a treatment effect wasevident (FIG. 5; P<0.025). Among treated groups, the response pattern ofmean fibre diameter was similar to that of the clean wool growth rate.By September, an increase of 2 μm in mean fibre diameter (P=0.05) wasobserved in the PRL-INF group. The mean fibre diameter appeared to belower (P<0.10) in the PRL-INJ group relative to ND-lambed controls.

Clean fleece production over the 6-month duration of the trial differedsignificantly between treatment groups (FIG. 5). Mean clean fleeceweight for PRL-INF ewes was greater than that of ND-lambed controls(1.63 vs. 1.23 kg, standard error of the mean±0.13 kg, P<0.01).

Prolactin receptor gene expression in the infused ewes was significantlyelevated during and after the 18 day prolactin infusion (P<0.01)compared to the other treatment groups (FIG. 6). The high level ofreceptor expression in infused ewes with respect to untreated ewes wassustained for two months April to June (P<0.05).

There were no significant differences measured between the remainingtreatment groups over the course of the trial.

Trial 4

Effect of Differing Periods of Prolactin Treatment on Long-Term WoolGrowth and Prolactin Receptor Gene Expression

Trial 4 was conducted to compare different periods of exogenousprolactin administration on wool growth.

Eighteen 3 year old Romney ewes were maintained indoors in individualpens under natural photoperiod for 12 months from March 2000. Data froma further 6 ewes were excluded on animal health grounds or because theyfailed to become pregnant. The ewes were fed to maintain a constantmaternal live weight (independent of conceptus and fleece weights). Acontrol group (ND non-pregnant) was not mated (n=6). Twelve other ewes(comprising Groups 2-4) were mated on 23 April.

An ovine prolactin pituitary extract (1.0 mg/kg/day) was administered byintravenous infusion via a jugular cannula commencing 11 days aftermating (4 May) for temporary periods of either 3 days (Group 2, n=6), 9days (Group 3, n=3) or 18 days (Group 4, n=3).

The sheep were shorn on entry to the experiment and again at theconclusion of the experiment in March 2001 and a mid-side wool samplewas measured for fibre diameter and yield. Wool samples were clippedfrom a standardised 100×100 mm mid-side patch on the right side of eachewe at monthly intervals to determine changes in seasonal clean woolproduction and mean fibre diameter. Wool growth and plasma prolactinconcentration were measured at regular intervals and the results wereplotted in FIGS. 7 and 8.

Six midside skin biopsies were collected at intervals between April andJune for measurement of prolactin receptor long form gene expression byreal-time PCR and the results are plotted in FIG. 9.

Results

Lambing occurred between 14 and 21 September. Clean patch wool growthrates declined in all groups between March and August indicative of thewinter decline. Prolactin profiles over the trial in the control andinfused groups are shown in FIG. 7.

Prior to the prolactin infusion, clean patch wool growth rate did notdiffer between groups. However, during May, a short-term treatmenteffect was evident with higher wool growth in the 9-day infused groupcompared to either the 3-day (P<0.01) or the 18-day (P<0.02) infusedgroups (FIG. 8). A significant treatment effect was also apparent inJuly with wool growth rate in the 9-day infused group higher than eitherof the other infused groups (P<0.01). This difference in growth ratepersisted until November.

Over the first 2 months of the experiment the mean fibre diameter ofwool from the 9-day group did not differ from the mean fibre diametersof the other prolactin infused groups. However, during June a treatmenteffect was evident, with the 9-day mean fibre diameter significantlyhigher than the 3-day infused group (FIG. 8; P<0.01).

In July, the mean fibre diameter of the 9-day infused group wasapproximately 3 μm greater than the other infused groups (3-day, P<0.01;18-day, P<0.025). Significant mean fibre diameter differences persisteduntil October. Total cumulative patch clean wool production over the12-month duration of the trial differed significantly between treatmentgroups (FIG. 8). Mean total patch weight for the 9-day infused ewes wasgreater than that of 3-day infused ewes (0.91 vs. 0.74 g, P<0.025) andof the 18-day infused ewes (0.91 vs. 0.66 g, P<0.01).

Prolactin receptor gene expression dropped significantly in all groupsexcept the 3-day group following the commencement of the prolactininfusions (FIG. 9). Following the termination of the infusions,prolactin receptor gene expression rose in all infused groups althoughthis was not statistically significant.

Trial 5

Effect of Two Separate Periods of Prolactin Treatment on PlasmaProlactin and Prolactin Receptor Gene Expression in Non-Pregnant Ewes

Trial 5 was conducted to examine two separated periods of exogenousprolactin administration on prolactin receptor gene expression in sheepskin over spring and early summer.

Twenty-one, mixed age, non-pregnant Romney ewes were maintained indoorsin individual pens from September 2001 until February 2002. The eweswere fed to maintain a constant live weight. They were divided into 3groups: short day controls (n=7), short day prolactin infused (n=7) andnormal day controls (n=7). An ovine prolactin pituitary extract (1.0mg/kg/day) was administered to each ewe in the short day infused groupby intravenous infusion via a jugular cannula commencing on 9 Octoberfor 9 days. A second 9-day prolactin infusion was administered to thesame ewes commencing on 20 November, 33 days after the end of the firstinfusion.

Plasma prolactin concentrations were measured at regular intervals andten midside skin biopsies were collected at intervals between Octoberand December 2001 for measurement of prolactin receptor long form geneexpression by real-time PCR. The results are plotted in FIG. 10.

Results

In the absence of exogenous administration, prolactin concentrationsremained low in both short day groups, but were elevated in the naturalday group due to exposure to long day late-spring and early-summerphotoperiod (FIG. 10). Intravenous prolactin increased prolactin levelsin treated ewes to approximately 500 ng/ml (P<0.001), which fell rapidlyto baselines levels on termination of the infusions. Prolactin receptorgene expression was elevated by prolactin at both the first (P<0.01) andat the second (P<0.02) infusions.

Trial 6

Effect of Prolactin Infusion on Prolactin Receptor Gene Expression inPregnant Ewes of Two Different Sheep Breeds

Trial 6 was conducted to examine the effects of exogenous prolactinadministration on prolactin receptor gene expression in pregnant andlactating ewes of two different sheep breeds.

Four groups, each comprised of seven 1-2 year old Romney or Wiltshireewes were shorn and maintained indoors in individual pens from Marchuntil November 2002. The animals were fed to maintain a constantmaternal live weight (independent of conceptus and fleece weights). Theewes were mated in March, and maintained in short days (8L:16D). Half ofthe ewes were cannulated and infusions of prolactin (40 mg/day) for 9days commenced one week after mating. Blood samples and skin biopsieswere collected from all ewes at intervals to monitor plasma prolactinand skin prolactin receptor gene expression respectively. The resultsare plotted in FIG. 11.

Results

Five ewes failed to become pregnant and their data are excluded from theanalysis.

Until close to parturition, prolactin levels remained low except duringprolactin administration when levels in infused ewes increased toapproximately 500 ng/ml (P<0.001). On termination of the infusions,prolactin concentrations shortly before parturition, circulatingprolactin increased and remained elevated during lactation in all ewes(P<0.001). Prolactin receptor gene expression was higher in Wiltshirethan in Romney ewes for the 5 skin samples collected between May andSeptember (P<0.05). Prolactin treatment of Wiltshire ewes in April wasassociated with an increase in prolactin receptor gene expression inSeptember (P<0.05) compared with non-treated Wiltshire ewes.

Trial 7

Effect of Prolactin Infusion on Prolactin Receptor Gene Expression inRabbit Tissues

The effect of exogenous ovine prolactin administration on prolactinreceptor gene expression in three different rabbit tissues was measured.

Two New Zealand White rabbits were maintained in cages indoors underambient conditions and, fed a diet of formulated rabbit pellets adlibitum with access to fresh tap water. At 9 months of age, subcutaneousimplants releasing 1.67 mg/day of bromocriptine over 60 days (Cat,Number SC-231; Innovative Research of America Inc.) were inserted underanaesthetic. At the same time, the rabbits received a slow release 2 mlosmotic pump (Alzet Model 2ML1; Alza Corporation, Palo Alto, Calif.,USA) delivering either saline or ovine prolactin (1 mg/kg/day). Therabbits were blood sampled from the ear on Days 0, 3 and 7. The bloodwas centrifuged and the plasma stored at −20 C. until radioimmunoassayfor ovine prolactin. At Day 7, the rabbits were euthanased with sodiumpentobarbitone. Liver, mammary gland and skin were dissected from eachrabbit and snap frozen in liquid nitrogen prior to assay by real-timePCR for prolactin receptor gene expression. The results are plotted inFIG. 12.

Results

Prolactin treatment was associated with elevated prolactin geneexpression in liver and skin, but not mammary gland, at 7 days (FIG.12).

Trial 8

Effect of Prolactin Infusion on Prolactin Receptor Gene Expression inMice Tissues

The effect of two exogenous ovine prolactin treatments on prolactinreceptor gene expression in mice mammary gland was examined.

Swiss female mice were maintained in cages at a constant temperature of22 C., under a photoperiod regime of 14 hours light: 10 hours dark, feda diet of formulated mouse pellets ad libitum with access to fresh tapwater. At 21 days of age, subcutaneous implants releasing 250 μg/day ofbromocriptine over 60 days (Cat. Number SC-231; Innovative Research ofAmerica Inc.) were inserted under anaesthetic. At the same time, themice received a slow release 100 μl osmotic pump (Alzet Model 1007D;Alza Corporation, Palo Alto, Calif.) delivering ovine prolactin (400μg/day) for either 3 or 7 days. Mice were sacrificed in groups of 3 atintervals before, during and after prolactin administration. They wereanaesthetised using CO₂ and blood sampled by heart puncture, prior toeuthanasia by cervical dislocation. The blood was centrifuged and theplasma stored at −20 C. by radioimmunoassay for ovine prolactin. Themammary glands were dissected from each mouse and snap frozen in liquidnitrogen prior to assay by real-time PCR for prolactin receptor geneexpression. The results are plotted in FIG. 13.

Results

Administration of ovine prolactin via osmotic pumps produced circulatinglevels in mice of 149 ng/ml (3-day pumps) and 294 ng/ml (7 day pumps).These differences were significant compared with saline treated controls(P<0.0.02, 3-day pumps; P<0.02, 7-day pumps). Prolactin receptor geneexpression was elevated in the 3-day infused group compared to both thecontrol group and the 7-day infused group at days 7 and 14 (P<0.05).

Discussion of Trials

Long-Term Regulation of Prolactin Receptor Gene Transcription byProlactin

The inventors have demonstrated that expression of the prolactinreceptor gene is regulated in the skin over a period of 3 months inresponse to a hormonal stimulus.

Earlier studies showed that changes in circulating prolactin couldtrigger hair cycle progression [Dicks et al., 1994; Pearson et al.,1993; Pearson et al., 1999a]. The close association between the level ofprolactin receptor mRNA in the skin and follicle growth status suggeststhat cellular activity in the follicle is related to receptor abundanceand the consequent level of signalling. Hence, prolactin may functionnot only to bind and activate its receptors but also to contribute toregulation of those receptors.

Both down- and up-regulation of prolactin receptor were evident in ovineskin over the 30 day period of steadily increasing plasma prolactin.During the first seven days of the induced wool growth cycle inWiltshire sheep (Trial 1), there was an initial decline in the abundanceof prolactin receptor mRNA associated with an increase in circulatingprolactin.

As plasma prolactin continued to increase, prolactin receptor regulationwas reversed and mRNA became more abundant in the skin. The concurrenceof peak prolactin receptor expression and high circulating prolactinwith the initiation of follicle growth suggests a stimulatory role infollicle recrudescence, as in mammary and reproductive tissues [Cassy etal., 1998].

Over short periods of time, such influences of prolactin on its ownreceptor are well recognised. Indeed, simultaneous and oppositeprolactin receptor changes have been shown in different organs of ratsinfused with ovine prolactin and growth hormone, depending onreproductive status and lactogen concentration [Barash et al., 1986].

Rapid “down-regulation” of binding sites has been attributed to anincrease in the rate of receptor internalisation and degradation [Djianeet al., 1979; Djiane et al., 1982]. However, long-term up-regulation ina seasonal animal in response to a prolactin surge has not previouslybeen demonstrated.

This allows for the possibility of enhancing or inhibiting physiologicalresponses to prolactin by appropriate priming treatments that changeprolactin receptor numbers in the target tissue. Such effects aredemonstrated in Trials 2 and 3 where large changes in wool growth weremeasured in response to alterations of prolactin status in prior months.In Trial 2, this could be seen in response to increased prolactin overpregnancy, parturition and lactation associated with long dayphotoperiod. In Trial 3, an 18-day infusion of prolactin immediatelyafter mating was associated with increased wool production duringpregnancy and after lambing.

Such long-term effects are also demonstrated in Trials 2-6 and in Trial8 where increased levels of prolactin receptor mRNA were observed duringand after a short-term prolactin treatment. Further, these effects aredependent on the prolactin profile (Trial 3), duration of infusion(Trial 4), timing of the infusion (Trial 5) and genotype (Trial 6).Treatments to alter future physiological responses to prolactin willvary with genotype, species and tissue (Trials 6, 7 and 8) but can beassessed on the basis of similar trials to determine prolactin receptorresponses to profiles of exogenous prolactin or of prolactin mimetics.To assist in such assessments, the inventors have developed amathematical model of the prolactin signalling system as described inthe following section.

Mathematical Model Including Equations and Diagram

The model structure is represented in FIG. 14. The variables of interestare the concentration of plasma prolactin and the numbers of unboundprolactin receptor, hormone-receptor dimers and hormone-receptor trimersat any time, t. Prolactin binding is sequential. First, the hormoneinteracts with its receptor through one binding site forming an inactivehormone-ligand complex. Then, prolactin binds to a second receptor,which leads to formation of signalling trimeric complex consisting of aprolactin molecule and its two receptors. We assume that the biologicaleffect of the hormone is a function of the trimer concentration.

In the model, the number of bound receptors increases due to binding ofprolactin to unbound receptors and decreases because of degradation anddissociation back to the unbound state. We assume the rate of bindingand dissociation obey a mass-action law and that the degradation ofreceptors is represented as a first order process.

Denoting the number of receptors in dimer and trimer complexes by B₁(t)and B₂(t) respectively, the number of unbound receptors by U(t) and theplasma prolactin concentration by P(t), we express this statementmathematically by the differential equation$\frac{\mathbb{d}B_{1}}{\mathbb{d}t} = {{\alpha\quad{P(t)}{U(t)}} - {\alpha_{1}{B_{1}(t)}{U(t)}} - {d\quad{B_{1}(t)}} + {d_{12}{B_{2}(t)}} - {\delta_{1b}{B_{1}(t)}}}$$\frac{\mathbb{d}B_{2}}{\mathbb{d}t} = {{\alpha_{1}{B_{1}(t)}{U(t)}} - {d_{12}{B_{2}(t)}} - {\delta_{2b}{B(t)}}}$

Here α and α₁ are rates of formation of dimer and trimer complexesrespectively and d, δ_(1b), d₁₂, δ_(2b) are the dissociation anddegradation rates of hormone-receptor complexes respectively.

The number of unbound receptors decreases because of binding anddegradation and increases due to synthesis and dissociation processes.We separate receptor synthesis at a constant rate, α, from inducedsynthesis with the rates depending on the size of the pool of signallingtrimer complex. In the current model, we take this dependence on B₂(t)to be Michaelis-Menten. Then it is linear for small values andsaturating for large values of B(t). The corresponding differentialequation for number of unbound receptors is$\frac{\mathbb{d}U}{\mathbb{d}t} = {a - {\delta_{u}{U(t)}} - {\alpha\quad{P(t)}{U(t)}} - {\alpha_{1}{B_{1}(t)}{U(t)}} + {d\quad{B_{1}(t)}} + {d_{12}{B_{2}(t)}} + \frac{\mu\quad{B_{2}(t)}}{b_{0} + {B_{2}(t)}}}$

The parameters μ and b₀ are associated with induced synthesis, with μbeing the maximum rate of induced synthesis and b₀ the number ofreceptors bound in trimer complexes at which the rate of inducedsynthesis is half the maximum value. The parameter δ_(u) is associatedwith the natural protein degradation.

The concentration of plasma prolactin decreases because of clearance,whilst secretion and artificial prolactin input cause an increase. Theseterms have already been described in the previous equations so thedifferential equation for concentration of plasma prolactin is$\frac{\mathbb{d}P}{\mathbb{d}t} = {{{- \gamma}\quad{P(t)}} + k_{0} + {k_{1}(t)}}$where γ is the clearance rate constant, k₀ and k(t) describe thesecretion and external input of prolactin respectively.

It is possible to parameterise the model for a particular species ortissue by setting the appropriate variables in the model to design theoptimal timing, plasma profile and dosage of a temporary sustainedprolactin (or a mimetic) treatment or immunological manipulation toalter short and long-term physiological responses to these.

For the simulations shown below we have taken values for parameters fromthe range currently available in literature [Gertler et al., 1996]α=4.15 (litres·nmol⁻¹·day⁻¹), α₁=3.02 (litres·nmol⁻¹·day⁻¹), d=12.96 (day⁻¹), d₁₂=4717.4 (day⁻¹),b₀=125000 (nmol), γ=35.6 (day⁻¹), μ=310000, δ_(1b)=δ_(b)=0.75 (day⁻¹),δ_(u)=0.53 (day⁻¹), and α=0.827.2 (nmol/day). At this stage, we ignoreseasonal and animal physiological effects and take prolactin secretionat the constant rate k₁=0.39 (nmol/day) in accordance with experimentalresults for sheep held in short days [Pearson et al., 1996; Nixon etal., 2002].

Initial conditions are determined by equilibrium under the aboveconditions. The number of unbound receptors drops as the prolactin isinfused, with a corresponding rise in bound receptor number. The inducedsynthesis of unbound receptors due to the presence of bound receptors(positive up-regulation) halts the decline, and once the infusion isswitched off the number of bound receptors declines back to the initialequilibrium, the number of unbound receptors reaches their highest leveland then relaxes to the equilibrium level (FIGS. 15 and 16).

FIG. 17 shows the simulation of two consequent nine-day prolactininfusions. FIG. 18 shows the comparison of effects of infusion andadministration of the same amount of prolactin by series of injections.

Discussion of Model

The inventors have devised the model to predict the effect of aparticular treatment as a guideline for developing the best method fortreatment or of experimental design.

Using this model, it is possible to parameterise for a particularspecies or tissue and design the optimal timing, plasma profile anddosage of a temporary sustained prolactin (or a mimetic) treatment orimmunological manipulation to alter short and long-term physiologicalresponses to these. The effect of prolactin treatment strongly dependson parameters of the model. These can vary significantly for differenttissues and species.

The five model simulations presented in FIGS. 15-19 illustrate how theinvention can be utilised and show how prolactin treatment in sheep (inthese examples presented as 9 day infusions or a series of 9 dailyinjections of exogenous prolactin) predicts short and long-term changesin the number of bound and unbound prolactin receptors. The simulationsshow:

Simulation 1 (FIG. 15): A single prolactin fusion of 9 days durationcommencing at day 5 causes an initial decrease in the number of unboundreceptors followed by a up-regulation in unbound receptor numbers (andtherefore increases potential capacity to respond to subsequentprolactin or prolactin mimetic stimulation). The up-regulation ofunbound receptors is in accord with the mRNA data from Trial 1 (FIG. 2).

Simulation 2 (FIG. 16): A single prolactin infusion commencing at day 17causes an initial decrease in the number of unbound receptors followedby a relatively long-term up-regulation in unbound receptor numbers, butdisplaced with respect to Simulation 1.

Simulation 3 (FIG. 17): An initial prolactin (or prolactin mimetic)treatment commencing at day 5 results in an augmentation of unboundreceptors at the time of a second treatment commencing at day 17 (andtherefore predicts an increase in any physiological response at thattime). This is consistent with the wool data presented in Trials 2 and 3(FIGS. 3 and 4).

Simulation 4 (FIG. 18): The effect of prolactin administration byinfusion is compared with the effect of series of prolactin injections.The results are consistent with Trial 3 (FIG. 4).

Simulation 5 (FIG. 19): The effect of prolactin infusion is sensitive tothe duration and amplitude of the injection. For the 9 days infusion,the concentrations of bound receptors were simulated over a wide rangeof infused hormone concentrations. Trimer concentration first increaseswith hormone concentration until a maximum is reached, then decreasescontinuously at higher prolactin concentrations. For the chosenparameterisation and duration of infusion, a maximum response is reachedfor a ten-fold increase in prolactin level. This value can be shifted tohigher concentrations of prolactin by increasing the infusion duration.The results are consistent with Trial 8 (FIG. 13).

Aspects of the present invention have been described by way of exampleonly and it should be appreciated that modifications and additions maybe made thereto without departing from the scope thereof.

REFERENCES

-   Akers R M, Bauman D E, Capuco A V, Goodman G T, Tucker H A (1981):    Prolactin regulation of milk secretion and biochemical    differentiation of mammary epithelial cells in periparturient cows.    Endocrinology 109:23-30.-   Anthony R V, Smith G W, Duong A, Pratt S L, Smith M F (1995): Two    forms of the prolactin receptor messenger ribonucleic acid are    present in ovine fetal liver and adult ovary. Endocrine 3:291-295.-   Aston R, Holder A T, Rathjen D A, Trigg T E, Moss B A, Pell J A    (1991): Antibody mediated enhancement of growth hormone activity:    application to animal production. Proceedings of the New Zealand    Society of Animal Production 51:227-234.-   Auldist M J, Walsh B J, Thomson N A (1998): Seasonal and lactational    influences on bovine milk composition in New Zealand. Journal of    Dairy Research 65:401-411.-   Barash I, Madar Z, Gertler A (1983): Down regulation of prolactin    receptors in the liver, mammary gland and kidney of female virgin    rat infused with ovine prolactin or human growth hormone.    Biochemical and Biophysical Research Communications 116:644-650.-   Barash I, Madar Z, Gertler A (1986): Short-term regulation of    prolactin receptors in the liver, mammary gland and kidney of    pregnant and lactating rats infused with ovine prolactin or human    growth hormone. Molecular and Cellular Endocrinology 46:235-244.-   Barenton B, Pelletier J (1980): Prolactin, testicular growth and LH    receptors in the ram following light and 2-Br-alpha-ergocryptine    (CB-154) treatments. Biology of Reproduction 22:781-790.-   Barrington G M, Besser T E, Gay C C, Davis W C, Reeves J J, McFadden    T B (1997): Effect of prolactin on in vitro expression of the bovine    mammary immunoglobulin G1 receptor. Journal of Dairy Science    80:94-100.-   Beattie J, Holder A T (1994): Location of an epitope defined by an    enhancing monoclonal antibody to growth hormone: some structural    details and biological implications. Molecular Endocrinology    8:1103-1110.-   Bole-Feysot C, Goffin V, Edery M, Binart N, Kelly P A (1998):    Prolactin (PRL) and its receptor: Actions, signal transduction    pathways and phenotypes observed in PRL receptor knockout mice.    Endocrine Reviews 19:225-268.-   Brinklow B R, Forbes J M (1983): Prolactin infusion causes increased    nitrogen retention in lambs in continuous darkness. Proceedings of    the Nutrition Society 42:38A.-   Cassy S, Charlier M, Belair L, Guillomot M, Charron G, Bloch B,    Djiane J (1998): Developmental expression and localization of the    prolactin receptor (PRL-R) gene in ewe mammary gland during    pregnancy and lactation: Estimation of the ratio of the two forms of    the PRL-R messenger ribonucleic acid. Biology of Reproduction    58:1290-1296.-   Cassy S, Charlier M, Guillomot M, Pessemesse L, Djiane J (1999):    Cellular localization and evolution of prolactin receptor mRNA in    ovine endometrium during pregnancy. FEBS Letters 445:207-211.-   Choy V J, Nixon A J, Pearson A J (1997): Distribution of prolactin    receptor immunoreactivity in ovine skin and during the wool follicle    growth cycle. Journal of Endocrinology 155:265-275.-   Craven A J, Ormandy C J, Robertson F G, Wilkins R J, Kelly P A,    Nixon A J, Pearson A J (2001) Prolactin signaling influences the    timing mechanism of the hair follicle: analysis of hair growth    cycles in prolactin receptor knockout mice. Endocrinology    142:2533-2539.-   Curlewis J D (1992): Seasonal prolactin secretion and its role in    seasonal reproduction: a review. Reproduction, Fertility and    Development 4:1-23.-   Dahl G E, Elsasser T H, Capuco A V, Erdman R A, Peters R R (1997):    Effects of a long daily photoperiod on milk yield and circulating    concentrations of insulin-like growth factor-1. Journal of Dairy    Science 80:2784-9.-   Dicks P, Russel A J F, Lincoln G A (1994): The role of prolactin in    the reactivation of hair follicles in relation to moulting in    cashmere goats. Journal of Endocrinology 143:441-448.-   Djiane J, Dusanter Fourt I, Katoh M, Kelly P A (1985): Biological    activities of binding site specific monoclonal antibodies to    prolactin receptors of rabbit mammary gland. Journal of Biological    Chemistry 260:11430-11435.-   Djiane J, Clauser H, Kelly P A (1979): Rapid down-regulation of    prolactin receptors in mammary gland and liver. Biochemical and    Biophysical Research Communications 90:1371-1378.

Djiane J, Delouis C, Kelly P A (1982): Prolactin receptor turnover inexplants of pseudopregnant rabbit mammary gland. Molecular and CellularEndocrinology 25:163-170.

-   Djiane J, Kelly P A (1993): Prolactin. In Thibault C, Levasseur M C,    Hunter R H F (eds): “Reproduction in mammals and man.” Paris:    Ellipses, pp 121-133.-   Fitzgerald B P, Evins J D, Cunningham F J (1981): Effect of TRH on    the secretion of prolactin in ewes at various stages of pregnancy    and in non-pregnant ewes during the breeding season and seasonal    anoestrus. Journal of Reproduction and Fertility 61:149-155.-   Forsyth I A, Lee P D (1993): Bromocriptine treatment of    periparturient goats long-term suppression of prolactin and lack of    effect on lactation. Journal of Dairy Research 60:307-317.-   Freeman M E, Kanyicska B, Lerant A, Nagy G (2000): Prolactin:    structure, function, and regulation of secretion. Physiological    Reviews 80:1523-1631.-   Freemark M, Driscoll P, Maaskant R, Petryk A & Kelly P A (1997)    Ontogenesis of prolactin receptors in the human fetus in early    gestation. Implications for tissue differentiation and development.    J Clin Invest 99: 1107-1117.-   Fulkerson W J, McDowell G H, Fell L R (1975): Artificial induction    of lactation in ewes: the role of prolactin. Australian Journal of    Bological Science 28:525-530.-   Gertler A, Grosclaude J, Strasburger C J, Nir S, Djiane J (1996):    Real time kinetic measurement of the interaction between lactogenic    hormones and prolactin-receptor extracellular domains from several    species support the model of hormone-induced transient receptor    dimerisation. Journal of Biological Chemistry 271:24482-24491.-   Goffin V, Binart N, Touraine P, Kelly P A (2002): Prolactin: the new    biology of an old hormone. Annual Review of Physiology 64:47-67.-   Grasselli F, Gaiani R, Tamanini C (1992): Seasonal variation in the    reproductive hormones of male goats. Acta Endocrinologica    126:271-275.-   Hansen P J (1985): Seasonal modulation of puberty and the    post-partum anestrus in cattle: a review. Livestock Production    Science 12:309-327.-   Hawker H, Crosbie S F, Thompson K F, McEwan J C (1984): Effects of    season on the wool growth response of Romney ewes to pasture    allowance. Animal Production in Australia 15:380-383.-   Hill R A, Pell J M (1998): Regulation of insulin like growth factor    I (IGF I) bioactivity in vivo: Further characterization of an IGF I    enhancing antibody. Endocrinology 139:1278-1287.-   Hogan B, Beddington R, Costantini F, Lacy E (1994) In: Manipulating    the mouse embryo: A laboratory manual, Second Edition, Cold Spring    Harbour Laboratory Press (Cold Spring Harbor, N.Y.), 487 pp.-   Holder A T, Aston R, Preece M A, Ivanyi J (1985): Monoclonal    antibody mediated enhancement of growth hormone activity in vivo.    Journal of Endocrinology 107:R9-R12.    Johke T, Hodate K (1983): Circulating prolactin leve and    lactogenesis in dairy cows. Proceedings of the Fifth World Congress    on Animal Production 2:251-252.-   Kelly P A, Robertson H A, Friesen H G (1974): Temporal pattern of    placental lactogen and progesterone secretion in sheep. Nature    248:435-437.-   Kendall P E (1999): Prolactin and wool growth in the Romney ewe:    Ph.D. Thesis. Palmerston North, NZ: Massey University.-   Lamming G E, Moseley S R, McNeilly J R (1974): Prolactin release in    the sheep. Journal of Reproduction and Fertility 40:151-168.-   L'Huillier P J, Soulier S, Stinnakre M-G, Lepourry L, Davis S R,    Mercier J-C, Vilotte J-L (1996): Efficient and specific    ribozyme-mediated reduction of bovine beta-lactalbumin expression in    double transgenic mice. Proceedings of the National Academy of    Sciences 93:6698-6703.-   Lincoln G A (1989): Significance of seasonal cycles in prolactin    secretion in male mammals. Perspectives in Andrology 53:299-306.-   Lincoln G A (1990): Correlation with changes in horns and pelage,    but not reproduction, of seasonal cycles in the secretion of    prolactin in rams of wild, feral and domesticated breeds of sheep.    Journal of Reproduction and Fertility 90:285-296.-   Lincoln G A, Ebling F J P (1985): Effect of constant-release    implants of melatonin on seasonal cycles in reproduction, prolactin    secretion and moulting in rams. Journal of Reproduction and    Fertility 73:241-253.-   Ling J K (1970): Pelage and molting in wild mammals with special    reference to aquatic forms. Quarterly Review of Biology 45:16-54.-   Loudon A S I, Brinklow B R (1990): Melatonin implants prevent the    onset of seasonal quiescence and suppress the release of prolactin    in response to a dopamine antagonist in the Bennett's wallaby    (Macopus rufogriseus rufogriseus). Journal of Reproduction and    Fertility 90:611-618.-   Maaskant R A, Bogic L V, Gilger S, Kelly P A & Bryant-Greenwood G    D (1996) The human prolactin receptor in the fetal membranes,    decidua, and placenta. J Clin Endocrinol Metab 81: 396-405.-   Maes M, Mommen K, Hendrickx D, Peeters D, D'Hondt P, Ranjan R, De    Meyer F, Scharpé S (1997): Components of biological variation,    including seasonality, in blood concentrations of TSH, TT3, FT4,    PRL, cortisol and testosterone in healthy volunteers. Clinical    Endocrinology (Oxford) 46:597-598.-   Martinet L, Ravault J P, Meunier M (1982): Seasonal variations in    mink (Mustela vison) plasma prolactin measured by heterologous    radioimmunoassay. General and Comparative Endocrinology 48:71-75.-   Masters D G, Stewart C A, Connell P J (1993): Changes in plasma    amino acid patterns and wool growth during late pregnancy and early    lactation in the ewe. Australian Journal of Agricultural Research    44:945-957.-   Mena F, Martinez-Escalera G, Aguayo D, Clapp C, Grosvenor C E    (1982): Latency and duration of the effects of bromocriptine and    prolactin on milk secretion in lactating rabbits. Journal of    Endocrinology 94:389-95.-   Michael S D (1976): Plasma prolactin and progesterone during the    estrous cycle in the mouse. Proceedings of the Society for    Experimental Biology and Medicine 153:254-257.-   Mondain-Monval M, Møller O M, Smith A J, McNeilly A S, Scholler R    (1985): Seasonal variations of plasma prolactin and LH    concentrations in the female blue fox (Alopex lagopus). Journal of    Reproduction and Fertility 74:439-448.-   Newbold J A, Chapin L T, Zinn S A, Tucker H A (1991): Effects of    photoperiod on mammary development and concentration of hormones in    serum of pregnant dairy heifers. Journal of Dairy Science    74:100-108.-   Nicoll C S (1980): Ontogeny and evolution of prolactin's functions.    Federation Proceedings 39:2563-2566.-   Nixon A J (1993): A method for determining the activity state of    hair follicles. Biotechnic and Histochemistry 68:316-325.-   Nixon A J, Choy V J, Parry A L, Pearson A J (1993): Fibre growth    initiation in hair follicles of goats treated with melatonin.    Journal of Experimental Zoology 267:47-56.-   Nixon A J, Ford C A, Oldham J M, Pearson A J (1997): Localization of    insulin-like growth factor receptors in skin follicles of sheep    (Ovis aries) and changes during an induced growth cycle. Comparative    Biochemistry and Physiology Part A 118A:1247-1257.-   Nixon A J, Ford C A, Wildermoth J E, Craven A J, Pearson A J (2002):    Regulation of prolactin receptor expression in ovine skin in    relation to circulating prolactin and wool follicle growth status.    Journal of Endocrinology 172:605-614.-   Oddy V H (1985): Wool growth of pregnant and lactating Merino ewes.    Journal of Agricultural Science 105:613-622.-   Ohlson D L, Spicer L J, Davis S L (1981): Use of active immunisation    against prolactin to study the influence of prolactin on growth and    reproduction in the ram. Journal of Animal Science 52:1350-1359.-   Ormandy C J, Binart N, Helloco C, Kelly, P A (1998): Mouse prolactin    receptor gene: genomic organization reveals alternative promoter    usage and generation of isoforms via alternative 3′-exon splicing.    DNA & Cell Biology. 17 (9): 761-770.-   Ostrom K M (1990): A review of the hormone prolactin during    lactation. Progress in Food and Nutrition Science 14:143.-   Parry A L, Nixon A J, Craven A J, Pearson A J (1996): The    microanatomy, cell replication, and keratin gene expression of hair    follicles during a photoperiod-induced growth cycle in sheep. Acta    Anatomica 154:283-299.-   Pearson A J, Ashby M G, Choy V J, Nixon A J, Wildermoth J E (1993):    The effects on wool follicle growth of suppression of the plasma    prolactin surge following short to long day transition. Journal of    Endocrinology 139:Abstract P46.-   Pearson A J, Ashby M G, Wildermoth J E, Craven A J, Nixon A J    (1999a): Effect of exogenous prolactin on the hair growth cycle.    Experimental Dermatology 8:358-360.-   Pearson A J, Kendall P E, Ashby M G, Wildermoth J E (1999b): Fleece    production patterns in Romney ewes: effects of photoperiod,    pregnancy and lactation. Proceedings of the New Zealand Society of    Animal Production 59:30-33.-   Pearson A J, Parry A L, Ashby M G, Choy V J, Wildermoth J E, Craven    A J (1996): Inhibitory effect of increased photoperiod on wool    follicle growth. Journal of Endocrinology 148:157-166.-   Peel C J, Taylor J W, Robinson I B, McGowan A A, Hooley R D, Findlay    J K (1978): The importance of prolactin and the milking stimulus in    the artificial induction of lactation in cows. Australian Journal of    Biological Sciences 31:187-195.-   Pell J M, Aston R (1995): Principles of immunomodulation. Livestock    Production Science 42:123-133.-   Peters R R, Chapin L T, Leining K B, Tucker H A (1978): Supplemental    lighting stimulates growth and lactation in cattle. Science    199:911-2.-   Peters R R, Chaplin L T, Emery R S, Tucker H A (1980): Growth and    hormonal responses to various photoperiods. Journal of Animal    Science 51:1148-1153.-   Peterson S W, Mackenzie D D S, McCutcheon S N (1990): Milk    production and plasma prolactin levels in spring- and autumn-lambing    ewes. Proceedings of the New Zealand Society of Animal Production    50:483-485.-   Peterson S W, Mackenzie D D S, McCutcheon S N (1991): Effect of    inhibiting prolactin secretion in ewes during late pregnancy on    lactogenesis. Proceedings of the New Zealand Society of    Endocrinology. Supplement to the Proceedings of the Endocrine    Society of Australia 34:199.-   Peterson S W, Mackenzie D D S, McCutcheon S N, Lapwood K R (1997):    Long-term bromocriptine treatment during late pregnancy has    differential effects on milk yields of single- and twin-bearing    ewes. New Zealand Journal of Agricultural Research 40:249-259.-   Petitclerc D, Vinet C, Roy G, Lacasse P (1998): Prepartum    photoperiod and melatonin feeding on milk production and prolactin    concentrations of dairy heifers and and cows. Journal of Dairy    Science 81:251.-   Posner B I, Kelly P A, Friesen H G (1975): Prolactin receptors in    rat liver: possible induction by prolactin. Science 188:57-59.-   Prandi A, Motta M, Chiesa F, Tamanini C (1988): Circannual rhythm of    plasma prolactin concentration in the goat. Animal Reproduction    Science 17:85-94.-   Regisford E G, Katz L S (1993): Effects of bromocriptine-induced    hypoprolactinaemia on gonadotrophin secretion and testicular    function in rams (Ovis aries) during two seasons. Journal of    Reproduction and Fertility 99:529-537.-   Regisford E G, Katz L S (1994): Testicular LH receptors are    dependent on serum prolactin (PRL) concentrations in rams. Biology    of Reproduction 50:112.-   Reiter R J (1980): The pineal and its hormones in the control of    reproduction in mammals. Endocrine Reviews 1: 109-131.-   Rhind S M, Robinson J J, Chesworth J M, Crofts R M J (1980): Effects    of season, lactation and plane of nutrition on prolactin    concentrations in ovine plasma and the role of prolactin in the    control of ewe fertility. Journal of Reproduction and Fertility    58:145-152.-   Rougeot J, Allain D, Martinet L (1984): Photoperiodic and hormonal    control of seasonal coat changes in mammals with special reference    to sheep and mink. Acta Zoologica Fennica 171:13-18.-   Royster M, Driscoll P, Kelly P A, Freemark M (1995): The prolactin    receptor in the fetal rat: cellular localization of messenger    ribonucleic acid, immunoreactive protein, and ligand-binding    activity and induction of expression in late gestation.    Endocrinology 136: 3892-3900.-   Rui H, Brekke I, Torjesen P A, Purvis K (1986): Homologous    up-regulation of the prolactin receptor in rat prostatic explants.    Molecular and Cellular Endocrinology 46:53-57.-   Schams D, Russe I, Schallenberger E, Prokopp S, Chan J S D (1984):    The role of steroid hormones, prolactin and placental lactogen on    mammary gland development in ewes and heifers. Journal of    Endocrinology 102:121-130.-   Schanbacher B D, Crouse J D (1980): Growth and performance of    growing-finishing lambs exposed to long or short photoperiods.    Journal of Animal Science 51:943-948.-   Smith A J, Mondain-Monval M, Møller O M, Scholler R, Hansson V    (1985): Seasonal variations of LH, prolactin, androstenedione,    testosterone and testicular FSH binding in the male blue fox (Alopex    lagopus). Journal of Reproduction and Fertility 74:449-458.-   Smith A J, Mondain-Monval M, Simon P, Andersen Berg K, Clausen O P    F, Hofmo P O, Scholler R (1987): Preliminary studies of the effects    of bromocriptine on testicular regression and the spring moult in a    seasonal breeder, the male blue fox (Alopex lagopus). Journal of    Reproduction and Fertility 81:517-524.-   Soares M J, Faria T N, Roby K F (1991): Pregnancy and the prolactin    family of hormones: Coordination of anterior pituitary, uterine, and    placental expression. Endocrine Reviews 12:402-423.-   Sumner R M W, Revfeim K J A (1973): Sources of variation and design    criteria for wool fibre diameter measurements for New Zealand Romney    sheep. New Zealand Journal of Agricultural Research 16:169-176.-   Tucker H A, Petitclerc D, Zinn S A (1984): The influence of    photoperiod on body weight gain, body composition, nutrient intake    and hormone secretion. Journal of Animal Science 59:1610-1620.-   Wettermann R P, Tucker H A (1974): Relationship of ambient    temperature to serum prolactin in heifers. Proceedings of the    Society for Experimental Biology and Medicine 146:908-911.-   Wettermann R P, Tucker H A, Beck T W, Meyerhoeffer D C (1982):    Influence of ambient temperature on prolactin concentrations in    serum of Holstein and Brahman×Hereford heifers. Journal of Animal    Science 55:391-394.-   Wong C C, Döhler K-D, Atkinson M J, Geerlings H, Hesch R-D, von zur    Mühlen A (1983): Circannual variations in serum concentrations of    pituitary, thyroid, parathyroid, gonadal and adrenal hormones in    male laboratory rats. Journal of Endocrinology 97:179-185.-   Woods J L, Orwin D F G (1988): Seasonal variations in the dimensions    of individual Romney wool fibres determined by rapid    autoradiographic technique. New Zealand Journal of Agricultural    Research 31:311-323.

Wu W X, Brooks J, Glasier A F, McNeilly A S (1995): The relationshipbetween decidualization and prolactin mRNA and production at differentstages of human pregnancy. Journal of Molecular Endocrinology 14:255-61.TABLE 1 Plasma prolactin concentrations in selected mammals MinimumMaximum Conc. Conc. Species and Breed Sex (ng/mL) Season (ng/mL) SeasonReference Mink M 4 late autumn to mid-winter 23 late spring [Martinet etal., 1982] F 6 late autumn to mid-winter 36 mid-spring [Martinet et al.,1982] Blue fox M <3 late summer to early spring 8 late spring [Smith etal., 1985] F <3 mid-summer to early spring 10 early summer[Mondain-Monval et al., 1985] Goat M <2 mid-winter 80 mid-summer[Grasselli et al., 1992] F <20 mid-winter >300 mid-summer [Prandi etal., 1988] Sheep Merino M <5 mid-winter 165 late spring [Lincoln, 1990]Romney F <10 mid-winter 200 early summer [Kendall, 1999] Wiltshire F <2mid-winter 150 late spring [Pearson et al., 1996] Mouse* F ˜50 [Michael,1976] Rat* M <20 [Wong et al., 1983] Human* M ˜11 [Maes et al., 1997] F˜24 [Maes et al., 1997]*non-seasonal, average value

TABLE 2 Oligonucleotide primers and probes used in PCR amplification ofprolactin receptor cDNA. A common forward primer was used in combinationwith different reverse primers to detect different prolactin receptorisoforms. Probes were labelled with a FAM reporter dye and a TAMRAquencher, according to the Taqman real-time PCR system (AppliedBiosystems) Amplicon size Oligonucleotide identity Primer sequence (bp)Mouse mPRLR common forward 5′-ATAAAAGGATTTGATACTCATCTGCTAGAG-3′ mPRLRlong form reverse 5′-TGTCATCCACTTCCAAGAACTCC-3′ 133 mPRLR short form 1reverse 5′-CATAAAAACTCAGTTGTTGGAATCTTCA-3′ 92 mPRLR short form 2 reverse5′-GGAAAAAGACATGGCAGAAACC-3′ 113 mPRLR short form 3 reverse5′-AGTTCCCCTTCATTGTCCAGTTT-3′ 113 mPRLR probe5′-CCCCCACTTCTGACTGTAGGACTTGC-3′ Sheep oPRLR forward5′-GCATGGTGACCTGCATCCT-3′ oPRLR long form reverse5′-CGGCTTGCCCTTCTCCA-3′ oPRLR probe 5′-CCACCAGTTCCAGGGCCAAAAAG-3′ RabbitrbPRLR forward 5′-GCAGTGGCTTTGAAGGGCTAT-3′ rbPRLR reverse5′-CCCAGGAACTGGTGGAAAGA-3′ 57 rbPRLR probe (minor groove5′-CATGGTGACCTGC-3′    binding design)

1-28. (canceled)
 29. A method for affecting a physiological response ofan animal to circulating levels of prolactin and/or prolactin mimetics,which comprises the step of: modulating prolactin receptors byeffectively signalling a sustained increase in the circulating level ofprolactin and/or prolactin mimetics, followed by a decrease back tonormal or low levels.
 30. A method as claimed in claim 1, wherein thesustained increase in circulating level of prolactin and/or prolactinmimetics is brought about by an increase in the photoperiod to which ananimal is exposed.
 31. A method as claimed in claim 1, wherein thesustained increase in circulating level of prolactin and/or prolactinmimetics is brought about by intravenous infusion.
 32. A method asclaimed in claim 1, wherein the sustained increase in circulating levelof prolactin and/or prolactin mimetics is brought about by a slowrelease bolus.
 33. A method as claimed in claim 1 wherein the sustainedincrease in circulating level of prolactin and/or prolactin mimetics isbrought about by way of an implant.
 34. A method as claimed by claim 29,wherein the reduction in circulating level of prolactin and/or prolactinmimetics is brought about by decreasing the photoperiod to which theanimal is exposed.
 35. A method as claimed in claim 29, wherein thedecrease in circulating level of prolactin and/or prolactin mimetics isbrought about by reducing or terminating the exogenous administration ofprolactin or prolactin mimetics.
 36. A method as claimed in claim 29,wherein the sustained increase is for a period of 3 to 18 days.
 37. Amethod as claimed in claim 36, wherein the sustained increase is for aperiod of 3 to 15 days.
 38. A method as claimed in claim 37, wherein thesustained prolactin increase is for a period of 3 to 9 days.
 39. Amethod as claimed in claim 29, wherein the circulating level ofprolactin is increased by 5 ng/ml to 800 ng/ml.
 40. A method as claimedin claim 39, wherein the circulating level of prolactin is increased by5 ng/ml to 200 ng/mg.
 41. A method as claimed in claim 29, wherein theincrease in the circulating level of prolactin is brought about by theincorporation into the animal's genome of an inducible recombinantnucleotide sequence encoding biologically active prolactin.
 42. A methodas claimed in claim 29, wherein the increase in the circulating level ofprolactin is brought about by the incorporation into the animal's genomeof a recombinant nucleotide sequence encoding a molecule which enhancesendogenous prolactin activity.
 43. A method as claimed in claim 41,wherein the recombinant nucleotide sequence is inserted into aninducible gene cassette under the control of a suitable promoter and/orenhancer sequence.
 44. A method as claimed in claim 42, wherein therecombinant nucleotide sequence is inserted into an inducible genecassette under the control of a suitable promoter and/or enhancersequence.
 45. A method as claimed in claim 43, wherein the promoter ismammary specific.
 46. A method as claimed in claim 43, wherein thepromoter is a milk protein.
 47. A method as claimed in claim 43, whereinthe promoter and/or enhancer sequence drives the transcription of therecombinant nucleotide sequence.
 48. A method as claimed in claim 41,wherein the recombinant nucleotide sequence contains 3′ flanking DNA tostabilize an mRNA.
 49. A method as claimed in claim 43, wherein the genecassette contains downstream regulatory sequences.
 50. A method asclaimed in claim 1, wherein the modulation of prolactin receptors isachieved by the administration of antibodies.
 51. An animal treated bythe method as claimed in claim
 1. 52. Animal products produced by ananimal treated by the method as claimed in claim 1.